Method for treatment of bacterial infections with once or twice-weekly administered rifalazil

ABSTRACT

A method for treatment of bacterial infections with rifalazil administered once-weekly or twice-weekly. A method for treatment of tuberculosis caused by  Mycobacterium tuberculosis , infections caused by  Mycobacterium avium  complex, infections caused by  Chlamydia pneumoniae  and infections caused by  Helicobacter pylori  by administering to a patient suffering from the bacterial infection 1-100 mg of rifalazil once or twice a week. In this dose regimen, the treatment is fast, efficacious and eliminates undesirable secondary symptoms observed with daily doses of 1-50 mg of rifalazil.

BACKGROUND OF THE INVENTION

This application is based on and claims priority of ProvisionalApplication Ser. No. 60/112,921 filed on Dec. 18, 1998.

FIELD OF THE INVENTION

The current invention concerns a method for treatment of bacterialinfections with rifalazil administered once-weekly or twice-weekly. Inparticular, the invention concerns a method for treatment oftuberculosis caused by Mycobacterium tuberculosis, infections caused byMycobacterium avium complex, infections caused by Chlamydia pneumoniaeand infections caused by Helicobacter pylori by administering to apatient suffering from the bacterial infection rifalazil once or twice aweek. In this dose regimen, the treatment is fast, efficacious andeliminates undesirable secondary symptoms observed with daily doses of1-50 mg of rifalazil.

BACKGROUND AND RELATED DISCLOSURES

Bacterial infection caused by mycobacterium species and similarinfections caused by Chlamydia pneumoniae or H. pylori cause serioushealth problems in the United States and worldwide. For example,tuberculosis, caused by Mycobacterium tuberculosis is one of the mostserious infectious diseases outside of developed countries, with overone billion people infected worldwide. The worldwide infection rateresults in eight million active tuberculosis cases annually and over twomillion deaths per year. In the United States, 26,000 near cases ofactive tuberculosis were reported in 1994. The number of active cases inthe United States is high because of the increase in patients with AIDSand the increase in immigration from developing countries. Moreover,there is reported an increase in multidrug resistance tuberculosis anddisseminated Mycobacterium avium complex infections.

Additionally, there is an increase in transmissible chlamydial diseasescaused by Chlamydia pneumoniae, nonmotile, obligate intracellulare tobacteria. Chlamydia pneumoniae causes respiratory infections, such aspneumonia, bronchitis, pharyngitis and sinusitis, and has beenassociated with about 10% of community-acquired pneumonia. The Chlamydiapneumoniae infections are geographically wide spread. Studies onantibody prevalence have shown that large number of people is infectedwith Chlamydia pneumoniae at one time or another.

Helicobacter pylori infections are infections of gastrointestinal tract.H. pylori is a gram-negative microphilic organism residing in humanstomach and intestine which is closely connected with acute gastritisand development of inflammation of mucous layer. Acute gastritis isassociated with epigastric pain, nausea and vomiting. The organism isdifficult to treat, delayed recurrences are frequent, and treatmentinvolves multiple antibiotic regimens.

It would thus be highly advantageous to provide a method for treatmentof the above-described diseases with new types of antibiotics which areable to efficaciously treat and/or eradicate the bacteria or organismscausing these diseases without necessity to utilize complex antibiotictreatments and, regimens which result in undesirable secondary symptomsand, adverse reactions.

It is, therefore, an object of this invention to provide a method fortreatment of Mycobacterium species, Chlamydia pneumoniae and H. pyloriinfections with once a week or twice a week administration of arelatively new antibiotic, rifalazil, that belongs to the class ofantibiotics called ansamycins. Rifalazil has the same or better activitythan either rifabutin or rifampin, the other two antibiotics of the sameclass and actively inhibits the growth of Mycobacterium tuberculosis,Mycobacterium avium species, Chlamydia pneumoniae and H. pylori whenadministered only once a week or twice a week in doses from 1 to 50 mg.Previously, rifalazil has been administered on daily basis and becauseof the severe secondary adverse reactions, was discontinued as a drugfor treatment of tuberculosis and other infection. Newly discoveredonce-week or twice-week regimen has the same efficacy as dailyadministration and yet eliminates or significantly decreases the adversereactions.

Rifalazil compound has been described in the U.S. Pat. No. 4,983,602where its antibacterial activity has been disclosed. Dosages describedin vitro and in mice animal models correspond to a dose from 10 mg to 10g/day for adults. However, when clinical trials with these doses of theantibiotic were administered daily, many adverse reactions occurred andthe treatment with rifalazil was discontinued.

All patents, patent applications and publications cited herein arehereby incorporated by reference.

SUMMARY OF THE INVENTION

One aspect of the current invention is a method for treatment ofbacterial infections with once or twice-week administration ofrifalazil.

Another aspect of the current invention is a method for treatment oftuberculosis with once or twice-week: administration of rifalazil.

Still another aspect of the current invention is a method for treatmentof Mycobacterium avium complex infections with once or twice-weekadministration of rifalazil.

Still another aspect of the current invention is a method for treatmentof Chlamydia pneumoniae infections with once or twice-weekadministration of rifalazil.

Yet another aspect of the current invention is a method for treatment ofHelicobacter pylori infections with once or twice-week administration ofrifalazil.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates decrease in white blood cells counts in daily dosingregimen used in a clinical trial on human volunteers where the dailydose of rifalazil was 5 or 25 mg compared to a control group receivingplacebo.

FIG. 2 illustrates decrease in white blood cells count in daily dosingregimen used in a clinical trial on human volunteers wherein the dailydose of rifalazil was 25 mg.

FIG. 3 illustrates decrease in white blood cells count in daily dosingregimen used in a clinical trial on human volunteers wherein the dailydose of rifalazil was 5 mg.

FIG. 4 illustrates changes in absolute neutrophil count in daily dosingregimen used in a clinical trial on human volunteers where the dailydose was 5 or 25 mg compared to a control group which received placebowithout rifalazil.

FIG. 5 illustrates decrease in absolute neutrophil count in daily dosingregimen used in a clinical trial on human volunteers wherein daily doseof rifalazil was 25 mg.

FIG. 6 illustrates decrease in absolute neutrophil count in daily dosingregimen used in a clinical trial on, human volunteers wherein daily doseof rifalazil was 5 mg.

FIG. 7 illustrates changes in platelets counts after 20 dailyadministration of 5 and 25 mg rifalazil to healthy volunteers in aclinical trial compared to a control group receiving placebo.

FIG. 8 illustrates changes in white blood cells after administration ofonce a week dose of 25 or 50 mg for five weeks to healthy volunteers ina clinical trial compared to a control group receiving placebo.

FIG. 9 illustrates changes in absolute neutrophil count afteradministration of once a week dose of 25 or 50 mg for five weeks tohealthy volunteers in a clinical trials compared to a control groupreceiving placebo.

FIG. 10 illustrates changes in platelet count after administration ofonce a week dose of 25 or 50 mg for five weeks to healthy volunteers ina clinical trial compared to a control group receiving solely a placebo.

DEFINITIONS

“Rifalazil” means3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxasinorifamycin also knownas KRM-1648.

“ANC” means absolute neutrophil count.

“ARS” means adverse reactions.

“AUCO₀₋₂₄” means area under the concentration time curve from 0 to 24hours.

“AUC_(0-∞)” means AUC from 0 hours to infinity.

“AZT” means azidodeoxythymidine.

“BID” means twice-a-day.

“BIW” means twice-a-week.

“BUN” means blood urea nitrogen.

“CFUs” means colony forming units.

“C_(max)” means maximum concentration.

“CL/F” means clearance (uncorrected for bioavailability).

“COSTART” means Coding Symbols for Thesaurus of Adverse Reaction Terms.

“CUM” means cubic micrometers.

“CYP” means cytochrome P450.

“ddC” means dideoxyctidine.

“EBA” means early bactericidal activity.

“EKG” means electrocardiogram.

“EMB” means ethambutol.

“G6PD” mean 5 glucose-6-phosphate dehydrogenase.

“GGT” means gamma glutamyl transpeptidase.

“IND” means Investigational New Drug.

“INH” means isoniazid.

“IP” means intraperitoneal.

“IRB” means Institutional Review Board.

“IV” or “i.v.” means intravenous administration.

“K_(e)” means terminal rate constant.

“K_(a)” means absorption rate constant.

“K/CU MM” means thousands per cubic millimeter.

“LDH” means lactic dehydrogenase.

“LEV” means level of loxacin.

“M/CU MM” millions per cubic millimeter.

“MAC” means Mycobacterial avium complex.

“MBC” means minimum bactericidal concentration.

“MIC” means minimum inhibitory concentration.

“MTB” means Mycobacterium tuberculosis.

“Mycobacterium avium complex” means Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium marinum, Mycobacteruim kansasii, andMycobacterium scrofulaceum.

“NADPH” means nicotinamideadeninedinucleotide hydrogen phosphate.

“PT” means prothrombin time.

“PTT” means partial thromboplastin time.

“PZA” means pyrazinamide.

“RBT” means rifabutin.

“Rif” or “RMP” means rifampin.

“QID” means once a day.

“QIW” means once-a-week.

“SD” means standard deviation.

“SGOT (AST)” means glutamic oxaloacetic transaminase (aspartatetransferase).

“SGPT (ALT)” means serum glutamic pyruvic transaminase, (alaninetransferase).

“TB” means tuberculosis.

“T_(max)” means time to maximum concentration.

“t_(½)” means terminal half-life.

“t_(½ abs)” means half-life of absorption.

“UUG” means micro-microgram.

“V_(beta)/F” means volume associated with terminal phase; (uncorrectedfor biovailability).

“V_(ss)/F” means volume of distribution at steady state (uncorrected forbioavailability).

“WBC” means white blood cell.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is based on findings and confirmation in vitro, invivo and in clinical trials that: once-a-week or twice-a-week doses of1-100, preferably 1-50 mg of rifalazil effectively treats bacterialinfection without adverse reactions and without undesirable secondarysymptoms observed with daily administration of this drug.

Although rifalazil was found to be effective against mycobacteriumspecies, it has never been used as a therapeutic agent for treatment ofmycobacterial diseases because at the daily dose regimen which wasthought to be necessary to its efficacious antibacterial activity,rifalazil caused severe adverse reactions and secondary symptoms. Theadverse reactions included flu-like symptoms with severe headache,malaise, fever, back pain, myalgia, chills, dizziness, nausea, vomiting,body pain and weakness. Additionally, the daily administration ofrifalazil resulted in changes in blood cell counts, particularly indecrease of white blood cells counts (leukopenia), absolute neutrophilcount and platelet count as well as in decreased blood hemoglobin. Forthese reasons, clinical studies involving daily dosing of rifalazil wereabandoned.

It has now been found and is a subject of this invention that rifalazilin once-a-week or at most twice-a-week dosing regimen is effective ineradication of Mycobacterium tuberculosis, Mycobacterium avium complex,Chlamydia pneumoniae and Helicobacter pylori and for treatment oftuberculosis, Mycobacterium avium infections, psittacosis,lymphogranuloma venereum, trachoma, inclusion conjunctivitis; caused byChlamydia pneumoniae and gastritis caused by H. pylori.

I. In Vitro and In Vivo Studies in Animal Models

Rifalazil and its related drugs rifampin and rifabutin, all belonging toa group collectively described as rifamycins, were known to exhibitantimicrobial activity against Mycobacterium species in vitro and invivo animal models.

A. Physical, Chemical and Pharmaceutical Properties of Rifalazil

Rifalazil is 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxasinorifamycin of the chemical structure

Rifalazil is a member of the rifamycins, a complex group of antibioticsoriginally isolated from Nocardia mediterranei that exhibitsantimicrobial activity against Mycobacteriun spp. The rifamycins belongto a class of antibiotics called. ansamycins, which contain macrocyclic,lactam bridges across non-adjacent (ansa) positions of an aromaticsystem.

Rifalazil is a nonpolar molecule that is stable and essentiallyinsoluble in water. Two chemically-related drug substances rifampin andrifabutin are known to have similar biological activity.

Rifalazil synthesis is disclosed in U.S. Pat. No. 4,983,602,incorporated herein by reference in its entirety. Its known in vitro andin vivo activity is described in Recent Res. Devel. Antimicrob. AgentsChemother., 2:37 (1997), aincorporated herein by reference. While thesestudies confirm the antibacterial activity of rifalazil in vivo as wellas in vitro, such activity is based on daily administration of 2.5 and 5mg of the drug to the mice infected with M. tuberculosis, correspondingto about 175 or 350 mg rifalazil dose/day/70 kg human.

Additionally, in vivo studies were performed where the therapeuticallyeffective doses of rifalazil and rifampin were given at variousintervals. When the dose 10 mg/kg (corresponding to 700 mg/70 kg human)six times a week was administered to treat M. tuberculosis infectioninduced in mice, there were no lesions observed in the lung and suchtreatment resulted in about 80% reduction of log CFU in lungs.Twice-a-week or once-a-week administration resulted in about 35%reduction of the bacteria and once-a-week administration of these veryhigh dosages resulted in about 42% reduction in CFU. However, the usedand documented doses were extremely and unphysioloqically high. Forhumans, the daily dose of rifalazil above 300 mg is unphysiological andeven 50 mg of rifalazil administered to humans daily causes severeadverse reactions.

B. Nonclinical Studies

Rifalazil was extensively tested in vitro and in vivo in animal modelsand compared to other ansamycins, rifampin and rifabutin. The followingis a brief description and summary of these studies.

1. Mechanism of Action

In vitro studies show that rifalazil acts on bacterial DNA-dependent RNApolymerase and inhibits the growth of aerobic and anaerobicgram-positive bacteria. However, rifalazil is relatively inactiveagainst gram-negativet bacteria. This spectrum of activity is similar torifampin and rifabutin, two related drugs.

Rifalazil is a potent inhibitor of many mycobacteriun Spp., includingthe M. tuberculosis (MTB) and M. avium complex (MAC), Chlamydiapneumoniae and H. pylori. Minimum inhibitory concentrations (MICs) ofrifampin for MTB range from <0.002 to 4.0 μg/mL, depending on the degreeof resistance to rifampin. When tested side by side against the samestrains, the activity of rifalazil in vitro is consistently greater thaneither rifabutin or rifampin. Minimum bactericidal concentrations (MBCs)are typically 2-4 fold higher than the MICs.

The efficacy of rifalazil have been examined in vivo in macrophage andin animal models. Rifalazil readily accumulates in human macrophages andis bactericidal at concentrations equivalent to the MBCs established invitro. In animal models of MTB infection, rifalazil was the most activesingle-agent against organisms in the spleen and lungs, although thecombination of rifalazil and isoniazid (INH) or rifalazil andpyrazinamide (PZA) was more effective against organisms in the lung thaneither drug alone (Antimicrobial Agents Chemotherapy, 40: 298 (1996)).

The therapeutic effects of rifalazil are also long-lasting. For example,in mice infected with M. intracellulare, rifalazil significantly reducedthe number of colony forming units (CFUs) in organs after four andeight. weeks of treatment and did so to a greater extent than rifabutinor rifampin. In a rabbit model of M. avium infection, rifalazil alsoreduced the bacterial load on organs compared to controls. Treatment ofMTB infection in mice with rifalazil and INH for 12 weeks completelysterilized the lungs and spleens of infected animals and eliminatedregrowth of the organisms for as long as 6 months post-treatment.

In chronic studies with dogs and rats, theno-observed-adverse-effect-level was 1000 mg/kg. The absolutebioavailability of ¹⁴C-rifalazil in rats at a dose of 3 mg/kg was 30 to40%, but was reduced at higher doses. Rifalazil was slowly eliminatedfrom the blood (mean terminal half-life of 12.5 hr) with a mean systemicclearance (CL/F) of 0.184 L/hr/kg in male rats and 0.217 L/hr/kg infemale rats. Significant partitioning of drug-related radioactivity tothe formed elements of the blood was observed. The principal routes ofelimination appeared to be hepatic metabolism with the majority (84%) ofthe radioactivity recovered in the feces. The urine contained a smallamount (5.5%) of radioactivity.

2. Rifalazil Antibacterial Activity in Vitro

The antimicrobial activity of rifalazil was measured in vitro against avariety of bacterial species. In vitro studies show that rifalazilinhibits the bacterial growth of aerobic and anaerobic gram-positivebacteria, but is relatively inactive against gram-negative bacteria.Rifalazil inhibits the growth of many Mycobacterium spp. (AntimicrobialAgents Chemotherapy, 35:542 (1991)), particularly the slower growingmycobacteria such as M. tuberculosis, M. avium, and M. intracellulare.Based on MIC₉₀ comparisons, as seen in Table 1, rifalazil was moreactive than rifampin.

TABLE 1 MIC₉₀ and Rifampin Against Mycobacterium spp MIC₉₀ (μg/mL)Species No. of Strains Rifalazil Rifampin M. intracellulare 31 0.1 12.5M. avium 18 1.56 100 M. tuberculosis 22 12.5 100 * MIC determined byagar dilution method. † RMP, rifampin.

The in vitro activity of rifalazil against M. tuberculosis has beendetermined by measuring the minimum inhibitory concentration (MIC) for avariety of clinical isolates and reference strains. The results of thesestudies are summarized in Table 2.

TABLE 2 Summary of In Vitro Susceptibility Studies for Rifalazil MICRange MIC₉₀ Ref. MIC Method No. Of Strains¹ (μg/mL) (μg/mL) 1 BACTEC 30(rif^(r) and rif^(s)) ≦0.002 to 4.0 2.0 2 BACTEC 24 (rif^(r) andrif^(s)) 0.0009 to 4.0 4.0 3 BACTEC 13 (rif^(s)) 0.0009 to 0.125 0.01564 BACTEC 11 (rif^(r)) 0.062 to 4.0 4.0 5 BACTEC 20 (rif^(s)) <0.125<0.125 6 BACTEC 20 (rif^(r)) <0.125 to >2.0 >2.0 7 7H11 agar 22 (rif^(r)and rif^(s)) ≦0.0125 to 12.5 12.5 8 7H11 agar 16 (rif^(s)) Not available≦0.0125 ¹rif^(r), rifampin-resistant; rif^(s), rifampin-sensitive.

Ref. 1: Antimicrobial Agents, Chemotherapy, 39: 2295 (1995); Ref. 2-5:Antimicrobial Agents, Chemotherapy, 39:440(1995), Ref. 7 and 8:Antimicrobial Agents. Chemotherapy, 35: 542 (1991).

As seen in Table 2, rifalazil is more active than 25rifampin based onMIC₉₀ comparisons, however, the spectra of its antibacterial activitiesare similar to rifampin.

The in vitro activity of rifalazil against M. tuberculosis has beendetermined by measuring the minimumi inhibitory concentration (MIC) fora variety of clinical isolates and reference strains. The results ofthese studies are summarized in Table 3.

Studies described in Antimicrobial Agents, Chemotherapy, 39: 2295,(1995) determined the MICs of rifalazil against thirty clinical isolatesand two stock cultures (H37Rv and Kurono) of M. tuberculosis (Table 3).

TABLE 3 MIC of Rifalazil, Rifabutin and Rifampin MIC (μg/mL)¹ forClinical Isolates² Reference M. MIC50 MIC90 tuberculosis strain Drug 50%inhibition 90% inhibition H37Rv Kurono Rifalazil 0.016 2.0 0.004 0.002Rifabutin 0.063 8.0 0.016 0.016 Rifampin 4.0 >128.0 0.125 0.063 ¹MICswere determined by BACTEC method. ²Thirty strains were tested.

Table 3 shows Minimum Inhibitory Concentrations (MICS) of rifalazil,rifabutin and rifampin for clinical isolates and two reference strainsof Mycobacterium tuberculosis.

As seen in Table 3, rifalazil had more than a 64-fold greater activitythan rifampin and a 4-fold greater, activity than rifabutin based oncomparisons of the MIC₅₀ and MIC₉₀. This increased activity of rifalazilwas also observed with the reference strains. An examination of theindividual MICs of the thirty isolates shows that rifalazil was moreactive than rifampin in all thirty isolates and more active thanrifabutin in twenty-eight isolates.

The MIC and NBC of rifalazil against extracellular M. tuberculosis andM. tuberculosis in human macrophages using strains H37Rv, Erdman, andAtencio were described in Antimicrobial Agents, Chemotherapy, 409:1482(1996). Extracellular and intracellular bacteria were exposed to varyingconcentrations of rifalazil for 7 or 8 days, macrophages were lysedwhere applicable, then the CFUs werea determined by plating on agar. TheMIC was defined as the 16 lowest concentration of rifalazil thatinhibited more than 99% of the growth following the drug-incubationperiod. The MBC was defined as the lowest concentration of rifalazilthat killed more than 99% of the bacteria following the drug-incubationperiod. The results of the study show that the MIC and MBC of rifalazilare at least 10-fold lower than rifampin for both intracellular andextracellular bacteria (Table 4).

TABLE 4 Minimum Inhibitory Concentration (MIC) and Minimum BactericidalConcentration (MBC) of Rifalazil and Rifampin (RMP) AgainstMycobacterium tuberculosis Strains Concentration (μg/mL) IntracellulareBacteria Extracellular Bacteria Rifalazil Rifampin Rifalazil RifampinStrain MIC MBC MIC MBC MIC MBC MIC MBC H37Rv 0.004 0.016 0.25 1.0 0.0080.031 0.12 0.5 Erdman 0.008 0.008 0.12 0.5 0.002 0.004 0.12 0.5 Atencio0.004 0.008 0.25 0.5 0.001 0.008 0.25 0.25

Antimicrobial Agent, Chemother., 40:1482 (1996).

3. Rifalazil Antibacterial Activity In Vivo

The therapeutic effect of rifalazil was examined by measuring gross lunglesions, bacterial loads, and survival time in mice infected with M.tuberculosis and subsequently treated with rifalazil or rifampin foreight weeks (Antimicrobial Agents, Chemotherapy, 39: 2295 (1995)). Ineach of these tests, rifalazil outperformed rifampin in treating thedisease.

The activity of rifalazil alone and in combination with other drugs inmice infected with the rifampin-sensitive M. tuberculosis strain Erdman(MIC_(rif)=0.06 kg/mL) was examined and results are described inAntimicrobial Agents Chemotherapy, 38: 2245 (1994). Treatment wasstarted one week after infection, and drug was administered five timesper week for four weeks. Control mice were sacrificed at the start oftreatment (early controls) and four weeks later (late controls).

Initial experiments compared the ability of rifalazil, rifabutin, orrifampin (all at 20 mg/kg) to reduce the bacterial load in lungs andspleens of infected mice compared to untreated mice. Rifalazil reducedbacterial loads to a significantly greater extent than the other twodrugs (P<0.01). No significant differences were observed betweenrifabutin and rifampin (P>0.05).

Additional experiments examined the ability of rifalazil (20 mg/kg)alone and in combination with INH (isoniazid, mg/kg), PZA (pyrazinamide,150 mg/kg), EMB (ethambutol, 125 mg/kg), or LEV (level of loxacin, 200mg/kg) to reduce this bacterial load in lungs and spleens of infectedmice.

Treatment with all drugs significantly reduced CFUs irn the spleencompared to controls, except for PZA. PZA did not significantly reduceCFUs compared to controls. Rifalazil was the most active single-agentagainst organisms in the spleen. Only the combination of rifalazil plusPZA was more active than rifalazil alone.

In lungs, treatment with rifalazil or INH significantly reduced cellcounts in lungs compared to early controls. Compared to late controls,treatment with rifalazil, INH, EMB, or LEV reduced cell counts in lungs.Rifalazil was the most active single-agent. The combinations ofrifalazil plus INH or rifalazil plus PZA were more active againstorganisms in lungs than treatment with rifalazil alone.

In a subsequent study, the treatment period was extended from 4 weeks to12 weeks and the regrowth of organisms in spleen and lung was measuredfor 24 weeks post-treatment. The combination of rifalazil (20 mg/kg) andINH (25 mg/kg) was significantly more effective at reducing the numberof CFUs in spleens and lungs of mice compared to rifalazil alone (20mg/kg), INH alone (25 mg/kg), rifampin alone (20 mg/kg), and thecombination of rifampin and INH (20 mg/kg and 25 mg/kg, respectively).

4. In Vitro and In Vivo Activity Against Other Bacteria

Rifalazil activity was also tested on other bacteria and organisms.Rifalazil shows a strong antibacterial activity against Chlamydiapneumoniae and against Helicobacter pylori.

Sensitivity testing was conducted in cell cultures3 against Chlamydiapneumoniae strain TW-1 83 using rifalazil, clanthromycin, orazithromycin. In these studies, rifalazil was 300-fold more potent thanclanthromycin and 1500-fold more potent than erythromycin. The in vivotesting of rifalazil used a mouse model infected with Chlamydiapneumoniae strain AR-39. The results showed that Chlamydia pneumoniaewas not detectable from the lungs of an animal five days after thecessation of rifalazil treatment by intraperitoneal injection ofrifalazil at 1 mg/kg QID for three days. All control animals remainedinfected.

Rifalazil bactericidal activities were also evaluated in vitro againsttwenty-four strains of Helicobacter pylori. In these studies, rifalazilexhibited more potent antimicrobial activities against Helicobacterpylori than amoxicillin and rifampin. Time-kill studies, described inAbstract, 4th Japan-Korea International Symposium on Microbiology,Takashimaya, Japan, Oct. 22-23 (1998), revealed that the CFUs at 24hours in the broth medium containing rifalazil at 0.04 mg/mL were morethan 4.5 log lower than the control at zero hours, indicatingrifalazil's potent bactericidal activity. Under the same conditionsamoxicillin at 0.31 mg/mL produced only 1 log decrease in CFU/mL after24 hours.

Results described above indicate that rifalazil has ver-y goodantibacterial activity and is a better choice of the drug for treatmentof bacterial infections caused by Chlamydia pneumoniae and Helicobacterpylori.

5. Pharmacology of Rifalazil

Pharmacological studies were undertaken in mice, rats, and dogs, and inisolated guinea pig ileum. The preclinical pharmacology data showed thatrifalazil has no important central/autonomic nervous system,respiratory, cardiovascular, digestive system, or renal pharmacologicaleffects.

Rifalazil had little effect on the clinical signs or general behavior ofmice following oral administration of 100, 300, or 1,000 mg/kg.Rifalazil had no effect on the spontaneous locomotor activity of mice at100 and 300 mg/kg. At 1000 mg/kg, rifalazil caused an increase inspontaneous locomotor activity for one hour.

6. Pharmacokinetics of Rifalazil

Pharmacokinetic parameters were determined following single and multipledoses in rats and dogs. The doses in rats and dogs were based upon thoseutilized in the single and multiple-dose toxicology studies. Inaddition, the absorption, distribution, metabolism, and elimination ofrifalazil was studied in rats and dogs. These studies confirmed priorfindings that there are species differences vis-a-vis sensitivity to andresponse to treatment with rifalazil.

Preclinical pharmacokinetic data in rats and dogs showed that thedisappearance of rifalazil and/or metabolites from whole blood is slowand that significant whole blood concentrations can be achievedfollowing repeated oral administration. Upon repeated dosing, a slightincrease in rifalazil C_(max) and AUC values was observed in rats anddogs. Such increase was consistent with the drug accumulation.Significant metabolism of rifalazil through deacetylation in both dogsand rats and hydroxylation in dogs only, occurred in both single andmultiple-dose studies. In addition, significant accumulation of bothmetabolites was observed following repeat rifalazil dosing in dogs.

7. Toxicology of Rifalazil

Single-dose and multiple-dose oral toxicology studies were conducted inmice, rats, and dogs. In addition, to maximize systemic exposure,single-dose intravenous (IV) and intraperitoneal (IP) studies wereconducted in rats and mice. Four-week and 13-week oral studies in dogsand rats and 26-week oral studies in rats included standard clinicalmeasurements and physical and pathological evaluations.

Under the conditions of these studies, rifalazil was relativelywell-tolerated in animal models following single or multiple-dose oraladministration. Hematological changes were noted following amultiple-dose oral administration at doses >30 mg/kg in both rats anddogs. Aside from the slight increase in liver weight in dogs at 1,000mg/kg, there was no evidence of any significant systemic target organchanges following four weeks of daily dosing in dogs and rats at dosesapproximately 100 times the highest projected clinical dose on a mg/kgbasis.

In the 26-week study, a dose-related lymphocytic depletion in thespleens of rats administered >30 mg/kg/day was observed. Lower absoluteand relative spleen weights in these animals correlated with thisapparent loss of lymphocytes. Lymphoid depletion in the spleens oftreated animals and decreased peripheral blood lymphocyte count showthat at certain concentration, rifalazil causes adverse reactions.However, there was no evidence that the animals in this study wereimmunosuppressed, as no opportunistic infection were observed.

The 13-week study of daily oral administration of rifalazil to dogsdemonstrated that the “no observable adverse effect level” wasconsidered to be 300 mg/kg for dogs. Lower lymphocytic counts were notreversed within four weeks after treatment, and therefore, the long-termconsequences of this effect in dogs are unknown.

These and other findings in mice demonstrate that there is clear speciesdifference in adverse reactions response between animals and humans.While in mice, rats, and dogs rifalazil dosages over 300 mg/kg were welltolerated in long-term studies, such tolerance was not found in humanvolunteers. The dose 300 mg/day caused severe adversie reactions anddaily dosing at this level could not be continued. The doses of 25, 50and 100 mg/day also caused severe adverse reactions in human volunteers.

The species differences between animals and human is therefore welldocumented and shows that the experimental results obtained inlaboratory animals cannot be extrapolated for humans.

II. Clinical Studies

Clinical studies were conducted both on healthy volunteers and onpatients diagnosed with tuberculosis.

A. Safety, Pharmacokinetics and Toxicity of Rifalazil in HealthyVolunteers

1. Clinical studies Design

A total of four clinical trials have been conducted to study the effectsof rifalazil in humans. Two single-dose Phase 1 clinical trials (001)and (002) assessed the safety and pharmacokinetics of rifalazil innormal, healthy, fasted subjects. In the 001 trial, a single 300 mg doseof rifalazil was administered to six subjects. In the 002 trial, singledoses of 30 mg or 100 mg were administered to eight subjects each. Theresults of these studies indicated a substantially higher incidence ofadverse reactions at the 300 mg dose compared to the 30 mg or 100 mgdose.

The third (003) and fourth (004) Phase 1 clinical trials weremultiple-dose studies. Because evidence from animal studies showedincreased bioavailability when rifalazil was administered with food, theclinical trials were designed to further assess the safety andpharmacokinetics of rifalazil in fed, normal, healthy subjects.

The third trial (003) was a randomized, rising, double-blind,multiple-dose, placebo-controlled study where subjects were observedduring the 14-consecutive days of drug administration in dosages 0, 5and 25 mg/day and for an additional 14 days without medication. Allsubjects received the dose within 30 minutes after eating a standardizedbreakfast. Subjects were divided into two groups. In Group 1, eightsubjects were randomized to a daily 25 mg dose oif rifalazil and foursubjects were randomized to placebo. Numerous adverse reactions began toappear with the 25 mg dose several days after dosing and it becameapparent that 25 mg daily dose was not safe. Consequently, after reviewof the safety data, the study protocol was amended to proceed with alower dose of 5 mg in group 2. Group 2 consisted of eight subjectsrandomized to a daily 5 mg dose of rifalazil and four subjectsrandomized to placebo.

The fourth trial (004) was a also a randomized, rising, double-blind,multiple-dose, placebo-controlled study. In this trial, weekly doses ofplacebo or rifalazil (25 mg or 50 mg) were administered to the subjectsfor a total of 4 weeks. All subjects received the dose within 30 minutesafter eating a standardized breakfast and were monitored for anadditional 14 days after the last dose. Four subjects were randomized toplacebo, six subjects to 25 mg rifalazil, and eight subjects to 50 mgrifalazil.

Both the third and fourth Phase 1 studies included a 14-day periodwithout medication following the last dose to observe the number andseverity of adverse reactions, and the time to resolution. Safety wasassessed by physical examination, monitoring vital signs and cardiacfunction, measurement of clinical laboratory values in blood, serum, andurine, and by documenting adverse reactions. Systemic drug levels weremeasured in each dose group.

2. Adverse Reactions Observed After Rifalazil Administration to HealthySubjects

Most of the adverse reactions in all four Phase 1 studies wereconsidered to be related to the study drug. The most common adversereaction in all studies was headache. Other commonly reported adversereactions, which collectively included “flu-like” symptoms, includedfever, back pain, chills, dizziness, nausea, vomiting, body pain, andweakness. These “flu-like” symptoms have been also described for twoother related rifamycins, rifampin and rifabutin.

Vital signs and physical assessments were within normal parameters anddid not change throughout any study. All EKG findings were coded asclinically insignificant and were normal, near-normal, or borderlinenormal.

3. Single-Dose Study—Clinical Trials 001 and 002

In the first (001) and second (002) clinical trials, adverse reactions,change in laboratory parameters and pharmacokinetic of rifalazil wereobserved in healthy volunteers receiving dosages of 30 mg, 100 mg and300 mg of rifalazil.

Results are seen in Table 5 which shows a comparative summary of thenumber of subjects reporting unique adverse reactions observed inclinical trials 001 and 002. As seen in Table 5 a substantially greaternumber of subjects reported adverse reactions with the 300 mg dosecompared to the 30 and 100 mg doses.

TABLE 5 Adverse Reactions in Healthy Volunteers Rifalazil Study 001 and001 002 002 Dose All Body Adverse 300 mg 0 mg 30 mg 100 mg Doses System¹Reactions n = 6 n = 9 n = 8 n = 8 n = 31 N of Subjects with any AE 4 2 31 10  N of Subjects with any AE 2 7 5 7 21  Body Abdominal Pain 1 0 0 01 Asthenia 2 0 0 1 3 Back Pain 2 0 0 0 2 Fever 3 0 0 1 4 Headache 3 0 31 4 Malaise 1 0 0 0 1 Pain 1 0 0 0 1 CV Tachycardia 3 0 0 0 3Vasodilation 0 1 1 1 3 DIG Abnormal Stools 1 0 0 0 1 Anorexia 1 0 0 0 1Dyspepsia 1 0 0 0 1 Vomiting 1 0 0 0 1 NER Dizziness 1 0 0 0 1Paraesthesia 1 0 0 0 1 Somnolence 1 0 0 0 1 RES Pharyngitis 2 0 0 0 2SKIN Dry Skin 1 0 0 0 1 Sweating 1 0 0 0 1 SS Taste Prevision 1 0 0 0 1¹BODY: body as a whole; CV: cardiovascular system; DIG: digestivesystem; NER: nervous system; RES: respiratory system; SKIN: skin andappendages; SS: special senses.

When the obtained adverse reactions were evaluated by severity, i.e.,mild adverse reactions, moderate reactions and severe reactions, as seenin Table 6, both the number and severity of drug-related adversereactions were found to increase with a dose. Regardless of the dose,all adverse reactions resolved within two weeks of drug discontinuation

TABLE 6 Number and Severity of Drug-Related Adverse Reactions in 001 and002 Clinical Trials N of Severity Study Dose subjects Mild ModerateSevere ARs 001 300 mg 6 41  8 4 53  002  0 mg 9 2 0 0 2  30 mg 8 4 0 0 4100 mg 8 5 2 0 7 TOTAL 31  52  10  4 66 

As seen in Table 6, 300 mg dose of rifalazil resulted in forty-one mild,eight moderate and four severe adverse reactions. In contrast, placebo,30 or 100 mg doses resulted in two, four or five mild adverse reactionsahd in zero, zero and two moderate adverse reactions, respectively. Nosevere adverse reactions were observed for placebo and for both 30 and100 mg dose groups.

In the clinical trials 001 and 002 all patients were submitted tolaboratory testing where the hematology, serum chemistry andurinanalysis were examined prior to dosing, during dosing and at least14 days following the cessation of drug administration.

In these two trials, white blood cell (WBC) counts, absolute neutrophilcounts (ANC), platelet counts, and blood hemoglobin were decreased in adose-dependent manner. These parameters returned to the normal rangewithin 14 days of final administration of rifalazil, and were noted tobe similar to effects produced by other rifamycins.

The pharmacokinetics of rifalazil in whole blood in these two clinicaltrials was similar to that of rifalazil pharmacokinetics in plasma.Converse to data generated from animal studies, human subjectsdemonstrated a higher (1.6:1) plasma to blood ratio. Therefore, futurepharmacokinetic analyses focused on rifalazil concentrations in plasma.Table 7 summarizes noncompartmental parameters derived from plasmaconcentrations in fasted subjects following administration of singledoses of 30 mg, 100 mg, or 300 mg of rifalazil in these studies.

TABLE 7 Comparison of Noncompartmental Pharmacokinetic ParametersDerived from Plasma Concentrations in Single Dose Studies 001 and 002Trial and Dose Parameters Rifalazil - 001 Rifalazil - 002 (mean) 300 mg100 mg 30 mg Tmax (h) 3.0 4.0 3.1 Cmax (ng/mL) 115.7 58.6 17.8 Half-life(h) distribution — — — t_(1/2) elimination 205.6 43.1 48.7 t_(1/2) AUC(ng.h/mL) 0-24 1454.6 800.4 196.9 0-∞ 4329.4 1543.3 504.9

4. Multiple-Dose Study—Clinical Trials 003 and 004

In the third (003) and fourth (004) clinical trials, adverse reactions,change in laboratory parameters and pharmacokinetics of rifalazil wereobserved.

A comparative summary of the number of subjects reporting unique adversereactions observed in clinical trails 003 and 004 appears in Table 8.

TABLE 8 Adverse Reactions in Single-Dose and Multiple Dose Trials StudyRifalazil-003 Rifalazil-004 Rifalazil-003/004 Body Adverse 5 mg/day 25mg/day 25 mg/wk 50 mg/wk 0 mg All Doses System¹ Reactions (n = 8) (n =8) (n = 6) (n = 8) (n = 13) (n = 43) N of Subjects with Any AE 8 8 5 8 635 N of Subjects with No AE 0 0 1 0 7 8 BODY Abdominal Pain 1 1 0 0 0 2Asthenia 0 3 2 2 1 8 Back Pain 6 7 3 4 0 20 Chills 2 4 3 5 0 14 Fever 26 4 7 1 20 Headache 5 8 4 7 3 27 Neck Pain 0 0 1 2 0 3 Pain 6 5 3 6 1 21CV Vasodilatation 0 3 0 3 1 7 DIG Anorexia 1 1 1 2 0 5 Diarrhea 1 2 0 00 3 Dry Mouth 1 1 0 0 0 2 Dyspepsia 1 1 0 1 1 4 Mouth Ulceration 0 0 1 00 1 Nausea 0 5 1 2 0 8 Thirst 0 0 1 1 0 2 Vomiting 0 2 1 2 0 5 MSArthralgia 1 0 0 0 0 1 Myalgia 0 3 0 1 0 4 NER Dizziness 0 4 3 3 1 11Insomnia 0 0 2 1 0 3 Somnolence 0 0 0 1 0 1 RES Pharyngitis 0 1 0 0 0 1Rhinitis 0 2 0 0 0 2 SKIN Pruritus 0 1 0 0 0 1 Dry Skin 0 0 1 0 0 1Sweating 0 1 0 1 0 2 SS Conjunctivitis 1 0 0 0 0 1 Eye Pain 2 3 0 0 0 5Taste Perversion 0 2 0 0 0 2 BODY: body as a whole; CV: cardiovascularsystem; DIG: digestive system; MS: musculo-skeletal system; NER: nervoussystem; RES: respiratory system; SKIN: skin and appendages; SS: specialsenses.

As seen in Table 8, more subjects reported adverse reactions withregimens of higher dose and/or shorter dosing intervals. Also, morefrequent episodes of the unique adverse reactions appeared with thehigher dose regimens.

Table 9 displays the most frequently reported adverse reactions whichwere reported 28 times in either study for 003 and 004 trials. All theseadverse reactions are considered “flu-like” symptoms.

TABLE 9 Adverse Reactions Observed in 003 and 004 Clinical Trials StudyRifalazil 003 Rifalazil 004 Adverse 0 mg 5 mg 25 mg 0 mg 25 mg 50 mgReactions (Placebo) /day /day All doses (Placebo) /wk /wk All DosesAsthenia 0 0 3 3 1 5 2 8 Chills 0 4 8 12 0 3 9 12 Dizziness 0 0 10 10 13 6 10 Fever 0 7 13 20 1 8 19 28 Headache 1 10 19 30 2 11 33 46 Nausea 00 8 8 0 1 4 5 Pain (back) 0 7 14 21 0 5 5 10 Pain (body) 0 2 5 7 1 3 1317 Pain (eye) 0 5 3 8 0 0 0 0 ¹Defined as >8 reports/event in eitherstudy. ²Pain terms not grouped to allow differentiation.

As shown in Table 9, for both daily multiple-dose 003 and once-a-weekmultiple dose 004 clinical trials, adverse reactions were dose dependentwith the highest incidence of fever, headache and back pain, observed inthe clinical trial 003 where the drug was administered daily. When themultiple-dose of rifalazil 25 mg/weekly was administered, the number ofadverse reactions in the same dose regimen (25 mg) decreasedsubstantially from thirteen to eight for fever, from nineteen to elevenfor headache and from fourteen to five for backpain.

These results clearly show that once a week dosage of rifalazil has muchlower incidence of adverse reactions.

In Tables 8 and 9, the clinical trial 003 are compared to clinical trial004, in terms of the adverse reactions associated with daily dosing ofrifalazil. In Tables 10 and 11, the number of drug-related adversereactions and severity of these reactions associated with daily dosingis shown vis-a-vis each subject and each dose for 003 clinical trial.

TABLE 10 Number of Adverse Reactions in 003 Clinical Trial Number ofGroup Dose Subject Number Drug-Related ARs 1 0 mg 01 0 (Placebo) 06 0 070 09 1 Total 1 1 25 mg/day 02 16 03 21 04 19 05 3 08 9 10 17 11 12 12 15Total 112 2 0 mg 01 0 (Placebo) 05 0 07 1 09 0 0 Total 1 2 5 mg/day 02 403 4 04 3 06 6 08 26 10 3 11 5 12 1 Total 52

As seen in Table 10, at daily dosing with 25 mg of rifalazil, subjectsexperienced total of one hundred and twelve adverse reactions while atthe daily dose of 5 mg, 8 subjects experienced total of fifty-twoadverse reactions. Placebo groups experienced only one adverse reactioneach. This study clearly show that multiple-dosages of rifalazil aredose dependent and that even a relatively small dosage of 5 mg ofrifalazil daily cause substantial increase in adverse reactions comparedto placebo.

Table 11 shows the number and severity of drug-relatecd adversereactions observed in the 003 clinical trial.

TABLE 11 Severity of Adverse Reactions in 003 Clinical Trial SeverityGroup Dose Mild Moderate 1 0 1 0 25 mg/day 102 10 2 0 1 0 2  5 mg/day 466 TOTAL 150 16

As seen in Table 11, severity of the adverse reactions was alsodose-dependent. When the dosage of 25 or 5 mg of rifalazil wasadministered daily, one hundred and two and forty-six mild adversereactions and ten and six moderate adverse reactions were observed. Withthe higher 25 mg/day dosage, the number of adverse reactions was morethan double for mild reactions and almost double for moderate reactions.

As seen in Tables 10 and 11, in Group 2 (5 mg/day), all eight subjectsreceiving drug reported at least one adverse reaction, compared to oneof four placebo subjects. By Day 7, five subjects continued to receiverifalazil while three subjects dropped from the study because of adversereactions. By Day 10, only one subject was still receiving drug. Dosingwas suspended after Day 11 by the site investigator. Dailyadministration of rifalazil was, therefore, found to be unacceptable tothe subjects and such daily administration had, to be discontinued.

Most adverse reactions observed in the 003 clinical trial werecategorized as mild, except for 16 events (16/166 or 9.6%) rated asmoderate as seen in Table 11. Ten of these moderate events occurredamong two subjects in Group 1 (25 mg/day). Seven of the eight subjectsreceiving drug discontinued the study due to experiencing a “flu-like”symptoms. Five of these subjects also had a concurrent, clinicallysignificant decrease in white blood cell count (WBC).

In Group 2 (5 mg/day), all eight subjects receiving drug reported atleast one adverse reaction compared to one of four placebo subjects(Table 10). The total number of adverse reactions reported was half theincidence of Group 1. In addition, the number of unique adversereactions experienced per patient was also about half the number inGroup 2 versus Group 1. Five of the eight subjects receiving rifalazilcompleted the study. Three subjects dropped due to adverse reaction. Onesubject experienced half of all the recorded adverse reactions for Group2 (26/52, see Table 10). This subject also experienced all of the sixadverse reactions that were graded moderate in severity within Group 2(Table 11). Although three of the eight subjects receiving rifalazilreported a mild, “flu-like” symptoms, only one of these subjectsdiscontinued the study early. All three subjects experiencing the“flu-like” symptoms had concurrent, clinically significant decreases inWBC.

In clinical trial 004, specifically, the adverse reactions associatedwith weekly dosing of rifalazil are listed in Tables 8 and 9, andcompared to 003 trial results. Tables 12 and 13 show the number andseverity of drug-related adverse reactions associated with once-a-weekadministration of rifalazil vis-a-vis each subject and each dose in 004clinical trial.

TABLE 12 Number of Adverse Reactions in 004 Clinical Trial Number ofDose Subject Number Drug-Related ARs 0 mg 02 2 (Placebo) 04 1 07 1 14 3Total 7 25 mg/wk 08 0 10 17 12 6 13 16 15 4 18 3 Total 46 5 mg/day 01 503 7 05 21 06 20 09 3 11 16 16 30 17 13 Total 115

As seen in Table 12, the number of adverse reactions observed followingonce-a-week administration of rifalazil to healthy volunteers wasdirectly related to the dosage of rifalazil administered. When thedosage was 25 mg/week, there were forty-six adverse reactions. When thedosage was 50 mg/week, then there were one hundred and fifteen adversereactions, almost 2.5 times more.

Table 13 shows a number and severity of drug-related adverse reactionsobserved in 004 clinical trial.

TABLE 13 Severity of Adverse Reactions in 004 Clinical Trial SeverityDose Mild Moderate Severe 0 mg (Placebo) 7 0 0 25 mg/wk 40 6 0 50 mg/wk93 21 1 TOTAL 140 27 1

Details of the adverse reactions associated with weekly dosing ofrifalazil appear in Tables 8, 9, 12 and 13. All eighteen subjectscompleted the 004 clinical trial. Fewer unique adverse reactions werereported per subject in the 25 mg group, versus the 50 mg group. Thereagain appeared to bet is a dose-related incidence of adverse reactions.More than twice the number of adverse reactions were reported bysubjects in the 50 mg group as compared to the 25 mg group (Table 12).Seven adverse reactions were reported by those receiving placebo. Onepatient in the 25 mg group reported no adverse reactions. The cluster ofreported adverse reactions remained similar to other studies. As seen inTable 13, most adverse reactions (140/168, 83%),were classified as mildin severity. Only six adverse reactions were rated as moderate in the 25mg group, compared to 21 moderate adverse reactions and one severeadverse reaction in the 50 mg group. One subject in the 50 mg groupexperienced 30 adverse reactions, twenty of which were graded as mild,nine moderate, and one severe.

Laboratory testing regimen for 003 and 004 trials was the same as for001 and 002 trials.

Abnormal laboratory values were observed at various times during both003 and 004 trials in three hematologic parameters: in platelet count,in white blood cells (WBC) counts, and in absolute neutrophil counts(ANC). Serum chemistry data revealed minor abnormalities in serumglucose, and liver function tests (SGOT, SGPT, GGT, and LDH) which werenot considered to be clinically relevant upon initial review. With theexception of elevated liver function tests in two subjects receivingplacebo in 003 clinical trial, all other laboratory measurement remainedwithin the normal established ranges. In clinical trial 003, allsubjects in Group 1 receiving 25 mg of rifalazil/day discontinued thestudy early.

The onset and magnitude of decrease in hematologic parameters appearedto be related to this dose. In order to better visualize the drop andsubsequent recovery in these parameters, the data were graphed tosynchronize subjects to their last dose. Results obtained from 001 and002 clinical trials are shown in FIGS. 1-7.

FIG. 1 shows mean plots of white blood cell (WBC) counts of healthyvolunteers receiving dosages 0 mg, 5 mg and 25 mg of rifalaziladministered daily. Normal range of white blood cell counts, shown inthe FIG. 1 as “L” and “H” lines, is between 4.5 and 11 K/CU MM. As seenin FIG. 1, the onset of decreases in the mean concentration of WBCs inboth groups appeared about 4 days after beginning of dosing at day −8,reaching the nadir about 1 to 2 days after the last dose. The WBC countsdecreased well below normal range of 4.5 K/CU MM.

FIG. 2 shows individual white blood cell counts in healthy volunteers(Group 1) receiving 25 mg of rifalazil daily for 14 days. As seen inFIG. 2, subjects in Group 1 experienced a larger drop in WBC counts,however, only one subject had the WBC counts that fell below2.0×10³/mm³, thle level which is approaching unsafe level.

FIG. 3 shows individual white blood cell counts in healthy volunteers(Group 2) receiving 5 mg of rifalazil daily for 14 days. As seen in FIG.3, subjects in Group 2 experienced lower decreases in WBC counts whichagrees with findings that the number and severity of adverse reactionare dose-dependent.

FIG. 4 shows mean absolute neutrophil count in twenty-four healthyvolunteers following administration of 0 mg, 5 mg and 25 mg of rifalazildaily for 14 days. As seen in FIG. 4, the absolute neutrophil countresults have shown less consistent patterns making it difficult toestablish the true nadir for each group.

FIGS. 5 and 6 show individual absolute neutrophil counts in Group 1receiving 25 mg of rifalazil daily for 14 days and Group 2 receiving 5mg rifalazil daily for 14 days, respectively. Four subjects in Group 1,receiving 25 mg of rifalazil, seen in FIG. 5, and 3 subjects in Group 2,receiving 5 mg of rifalazil, seen in FIG. 6, experienced ANC values<2.0×10³/mm³, however no ANC value fell below <1.0×10³/mm³ for anyindividual subject.

Platelet counts, shown in FIG. 7, demonstrated small changes relative toplacebo, with fewer changes occurring in the group receiving 5 mg ofrifalazil versus the group receiving 25 mg. All hematologic parametersreturned to normal within 14 days following administration of the lastdose.

Results obtained in 004 clinical trial where the healthy volunteersreceived only one dose a week for four weeks are seen in FIGS. 8-10.Consistent with prior observations in 001 and 002 clinical trials,overall, this study resulted in changes of hematologic parameters ofmuch smaller magnitude compared to the daily multiple-dose clinicaltrial 003.

FIG. 8 shows mean white blood cell plots for once a week dosage of 0 mg(control), 25 mg and 50 mg of rifalazil for four weeks. The subjects'hematological parameters were followed for an additional two weeks up today 36.

When the results seen in FIG. 8 (once-a-week administration of 50 and 25mg rifalazil) are compared to results seen in FIG. 1 (once-a-dayadministration of 5 and 25 mg of rifalazil), the differences in WBCcounts are readily observed. In FIG. 1, both 50 and 25 dosages showcontinuous drop in WBC counts to around 2.0 K/CU MM. In FIG. 8, on theother hand, WBC counts stayed overall within the normal range between4.5 and 11.0 K/CU MM.

FIG. 9 shows mean absolute neutrophil counts for once-a-week dosage of 0mg (control), 25 mg and 50 mg of rifalazil administered for four weeks.As above, subjects ANC were followed up to day 36. Three subjects in the25 mg group and four subjects in the 50 mg group had shown decreases inthe ANC to below 2.0×10³/mm³ (data not shown). However, no subjectexperienced a drop in ANC below 1.0×10³/mm³ which is considered unsafe.

FIG. 10 shows mean platelet plots for once-a-week dosage of 0 mg(control), 25 mg and 50 mg of rifalazil for four weeks with follow up today 36. As seen in FIG. 10, once a week dosages of rifalazil onplatelets were unremarkable without any observable changes outside ofthe normal range 150-450 K/CU MM.

All hematologic parameters returned to normal by 14 days, afteradministration of the last dose.

Pharmacokinetic analyses associated with clinical trials involvedmeasurement of concentrations of rifalazil in plasma and/or whole bloodof subjects participating in the four Phase 1 studies using highperformance liquid chromatography. Most pharmacokinetic parametersderived in these studies are reflective of different dosing schedules,and conditions, such as fasted or fed subjects, and thus are notdirectly comparable across studies.

Results are shown in Table 7 for the 001 and 002 clinical trials, inTable 14 for the 003 clinical trial and in Table 15 for the 004 clinicaltrial 004. Several pharmacokinetic patterns were consistently observed.Rifalazil appears to be slowly absorbed, widely distributed, and slowlyeliminated via a multi-phasic process. Inter-patient variability wasdemonstrated.

Due to extremely low levels of rifalazil measured in the urine,elimination of rifalazil seems to be non-renal, and probably occurs bythe fecal route. In addition, low levels of oxidative metabolites ofrifalazil were found in plasma. This further suggests that drug isexcreted in the feces either in unchanged form or as conjugates of theparent drug.

Attempts were made to analyze the multiple-dose pharmacokinetics using alinear 3-compartment model, and a 3-compartment model incorporatingenzyme induction. However, neither multi-compartmental model accuratelycharacterized the pharmacokinetic parameters in all subjects. Dailydosing and related early terminations due to adverse reactions,differences in bioavailability from a food effect, and nonconnectivedose administration further complicated these analyses. Therefore, onlynoncompartmental pharmacokinetic parameters were derived for clinicaltrials 003 and 004.

TABLE 14 Pharmacokinetics Parameters in 003 Clinical Trial Group 2 Group2 5 mg/day Group 1 Parameters 5 mg/day Last Dose 25 mg/day (mean) FirstDose (day 14) First Dose Tmax (h) 5.25 4.57 5.62 Cmax (ng/mL) 13.3 13.4141.26 Half-Life (h) distribution _(t1/2) 10.07 17.26 12.5 elimination_(t1/2) — — AUC (ng.h/mL) 0-24 137 187 479 0-∞ 178 — 688 ¹Steady-statenoncompartmental pharmacokinetics could not be assessed in Group 1 dueto early terminations and missed doses.

Table 14 shows noncompartmental pharmacokinetics parameters derived fromdaily dosing for up to 14 days in 003 clinical trial.

For the 004 clinical trial, the pharmacokinetic analysis of thismultiple-dose study was limited to a comparison of the noncompartmentalparameters obtained from the first dose (Day 1) data versus the fourth(last) dose (Day 22). Because of extensive sampling after the fourthdose, this study yielded the most complete data about terminalelimination of rifalazil given in a multiple-dose regimen. Results areshown in Table 15.

TABLE 15 Pharmacokinetic Parameters in 004 Clinical Trial DoseParameters 25 mg/wk 25 mg/wk 50 mg/wk 50 mg/wk (mean) First Dose LastDose First Dose Last Dose Tmax (h) 5.7 5.7 5.5 6.0 Cmax (ng/mL) 39.343.8 69.78 79.1 Half-Life (h) distribution _(t1/2) 10.5 11.3 9.5 12.0elimination _(t1/2) — 60.9 — 109.1 AUC (ng.h/mL) 0-24 472.2 552.6 795.31013.0 0-∞ 628.7 1347.3 1019.1 2840.6

As shown in Table 15, C_(max) and AUC values demonstratedose-proportionate increases. The C_(max) from the fourth dose (Day 22)was about 44 ng/mL in the 25 mg dose group, and about 79 ng/mL in the 50mg group. The change between values on Day 1 and Day 22 was notstatistically significant. The mean apparent terminal half-life wasapproximately 61 hours after the 25 mg dose, and about 110 hours afterthe 50 mg dose, although the range was quite variable (29.5 to 106.8hours and 65.9 to 148 hours, respectively). The AUC(₀₋₂₄) increased onDay 22 approximately 17% for the 25 mg dose and 27% for the 50 mg dose,relative to Day 1 AUC values. Similar noncompartmental pharmacokineticparameters were demonstrated after 25 mg doses in both 003 and 004clinical trials.

4. Summary and Conclusions of Phase I Clinical Trials 001-004

The four Phase I clinical trials have investigated the safety profileand pharmacokinetics in healthy male subjects following theadministration of rifalazil as single doses (300 mg, 100 mg, 30 mg),daily doses (25 mg, 5 mg) administered for 14 days, and weekly doses (50mg, 25 mg) administered for 4 weeks. Results demonstrated a cleardose-related incidence in the number and severity of adverse reactions.The predominate adverse reactions comprised “flu-like” symptoms, whichare also known to be present with rifampin and rifabutin, other agentsin the same pharmacologic class. The symptoms are generally mild, andrarely resulted in discontinuation of drug at lower and less frequentdosing regimens. For daily dosing regimen, however, the symptoms becamemore pronounced and several patients discontinued the study.

The changes in hematologic parameters also appeared to be dose-related.Although decreases in WBC and ANC values to less than the normal rangesoccurred, they returned to normal levels within two weeks of dosecompletion. Increasing the dosing interval by changing theadministration from once daily to once weekly diminished both the numberand severity of adverse reactions and also the decreases in hematologicparameters. In terms of frequency and severity, a dose of 25 mg or 50 mggiven weekly in healthy subjects yielded a tolerable safety profile.

Pharmacokinetic analysis has clearly demonstrated that theadministration of food with rifalazil delayed absorption and increasedC_(max) and AUC in a dose-proportional manner. The mean terminalhalf-life seen with the 25 mg dose was about 61 hours. Accumulation ofrifalazil with either 25 mg or 50 mg doses, given once weekly over 4weeks to healthy subjects, appeared to be minimal.

After administration of a dose of 25 mg, the average C_(max) achievedwas about 44 ng/mL, which was 2 to 3 times the M1C₉₀ ofrifampin-sensitive Mycobacterium tuberculosis (15.6 ng/mL) Furthermore,because of the partitioning of rifalazil into macrophages,therapeutically beneficial concentrations of rifalazil are expected topersist in macrophages longer than in plasma. Thus, plasmaconcentrations that fall below the MIC₉₀ during the dosing interval maybe clinically insignificant in disease treatment, because the drug maybe stored in macrophages.

B. Efficacy of Rifalazil Treatment in TB Patients—Clinical Trial 005

Following the studies performed on healthy volunteers, clinical trial005 was initiated for treatment of tuberculosis patients with isoniazid(INH) alone administered daily, or isoniazid combined with rifampin bothadministered daily, or isoniazid administered daily with either 10 mg or25 mg of rifalazil administered weekly.

Dosages of isoniazid and rifampin were adjusted to body weight ofpatients. When the patient's body weight was less than 35 kg, isoniazidwas administered in 10 mg/kg/dose with maximum dose 300 mg, and rifampinwas administered in 10 mg/kg/dose with maximum dose of 450 mg/dose. Whenthe patient's body weight was between 35 and 45 kg, isoniazid wasadministered in 300 mg/dose and rifampin in 450 mg/dose. When thepatient's body weight was greater than 45 kg, isoniazid was administeredas 600 mg/dose.

In these studies, the following tests were performed and are presentedin Tables 16-21. Change in Log₁₀ of colony forming units (CFU) per mL ofsputum of microbiologically evaluable patients is shown in Table 16.WBC, ANC and platelets counts in INH treated patients, is shown in Table17. WBC, ANC and platelet counts in patients treated with INH andrifampin, are shown in Table 18. WBC, ANC and platelet counts inpatients treated with INH daily and 10/25 mg of rifalazil weekly, areshown in Tables 19 and 20, respectively. Rifalazil concentration inpatients treated with INH and 10 or 25 mg of rifalazil, are shown inTable 21. Definite diagnosis and evaluation of treatment efficacyrequires direct examination of sputum for the presence of M.tuberculosis. The extent of M. tuberculosis infection is determined bynumber of bacteria able to form colonies and is expressed as colonyforming units. When the number of colony forming units (CFU) is high,the infection is unhindered. When the CFU decreases, the number ofactive M. tuberculosis bacteria decreases. The CFU, typically expressedas Log₁₀, have therefore important diagnostic and treatment evaluativevalue.

One challenge to evaluating the effectiveness of new diagnostics andtreatment for tuberculosis is the long time interval required formeasuring decreases in CFU levels and observable clinical improvements.That is to say, the treatment of tuberculosis is of long duration(typically at least 6 months) and as a consequence only after severalmonths of therapy is it possible to determine if clinical benefit isachieved. Therefore, clinical studies have been designated to evaluatethe early bactericidal activity (EBA) of new tuberculosis therapies. TheEBA of a therapy can be defined as the activity observed during theinitial phase (e.g., initial 1-2 weeks) of treatment. Clinical studiesthat incorporate EBA analyses are based on the contention that EBA is agood index of ultimate therapeutic benefit that is observed severalmonths later.

Table 16 which shows sputum baseline to day 15 in Log₁₀ CFU/mL of sputummicrobiologically evaluable patients compares the reduction in colonyforming units (CFU) for the four different groups of patients over thefirst two weeks of the therapy. The four groups of patients were treatedas follows: Group 1 received INH daily for 14 days, Group 2 received INHdaily plus rifampin daily for 14 days, Group 3 received INH daily for 14days plus rifalazil once per week (10 mg on day 1 and day 8) over 14days, and Group 4 received INH daily for 14 days plus rifalazil once perweek (25 mg on day 1 and day 8) over 14 days. Dosages of isoniazid andrifampin depended on the patient's weight and were as above. The numberof patients in each study group are presented as the N value.

The number of patients in Groups 1-4, was six, four, six, and six,respectively. The mean change in sputum CFU following these treatmentsis also shown. The change in CFUs is greatest for Groups 2 and 4 whichreceived daily treatment with INH in combination with rifampinadministered daily (Group 2) or INH administered daily in combinationwith rifalazil administered once-a-week at 25 mg dosages (Group 4).These data show that rifalazil administered weekly or twice weekly inrelatively very low dosages of 10 or 25 mg is an effective substitutefor rifampin (600 mg/day administered daily) in the routine therapy fortuberculosis.

TABLE 16 Change from Sputum Baseline to Day 15 in Log₁₀ CFU/mL of SputumMicrobiologically Valuable Patients Treatment Group INH + RMP INH +Rifalazil INH + Rifalazil Log₁₀ INH 400 mg + 400 mg + 400 mg + CFU/mL400 mg 600 mg 10 mg 25 mg N 6 4 6 6 Mean −1.58 (0.51) −2.61 (0.94) −2.22(0.98) 2.74 (0.73) (SD) Med −1.63 −2.84 −2.25 −2.78 Min −2.28 −3.41−3.24 −3.67 Max −0.92 −1.34 −0.65 −1.55

These results clearly show that administration of INH-rifalazilonce-a-week in 10 or particularly 25 mg doses is as efficacioustreatment for tuberculosis as treatment with INH-rifampin daily.

The four tables 17-20 show hematology data which are separated accordingto Groups 1-4. The group treatment corresponds to those seen in Table16. These data provide safety information regarding the adversereactions because hematologic perturbations are considered to be amongthe most clinically important adverse reactions associated withtreatment by rifamycin type drugs. In particular, the ANC value isconsidered to be a very meaningful safety index as it directly relatesto a patient's ability to fight infection.

As seen in Tables 17-20, in the upper section, white blood cell countsdecreased slightly after the baseline values in all groups during thetreatment but did not reach critically low levels. Once a week treatmentwith 10 mg rifalazil combined with 400 mg or less of INH administereddaily did not lead to decrease in WBC.

Upon comparison of the ANC values for Groups 1, 2, 3 and 4 (Tables17-20) a drop in ANC was observed after the baseline values for groups2-4. A lower ANC value, however, is not of great concern, however, untilit drops below 1.0 K/CU MM. That level was reached in only one patientin Group 3, treated with INH plus rifalazil at 10 mg but that patienthad a low ANC value to begin with.

Platelet levels were not affected by any treatment and were, therefore,not safety concern.

The important conclusions derived from the hematologic data is thatrifalazil does not cause a greater level of hematologic disturbances(safety concerns) than rifampin which is routinely used for treatment ofTB. Rifalazil is therefore as safe as rifampin and as efficacious inlower and less frequent dosages.

TABLE 17 WBC, ANC, and Platelet Counts (K/CU MM) - INH Baseline Day 4Day 8 Day 11 Day 15 Day 28 Day 42 n 8 8 7 7 7 3 2 WBC (K/CU UM) Mean(SD) 8.90 (1.87) 10.88 9.08 8.28 9.23 8.31 7.98 (6.87) (2.23) (0.97)(2.28) (2.48) (1.77) Med 8.55 8.81 8.63 8.10 8.49 7.06 7.98 Min 6.776.37 6.21 7.27 6.82 6.70 6.73 Max 11.60 27.19 12.06 10.03 12.40 11.169.23 ANC (K/CU UM) Mean (SD) 6.04 (1.92) 7.62 5.96 5.29 5.95 5.63 6.96(6.29) (2.05) (0.96) (2.08) (1.95) (0.34) Med 5.68 5.47 5.22 5.06 6.044.60 6.96 Min 3.90 3.79 3.34 4.17 3.51 4.41 6.72 Max 8.74 22.51 9.326.57 8.84 7.87 7.2 Platelets (K/CU UM) Mean (SD) 295.75 309.38 310.13336.10 298.59 287.27 297.10 (93.28) (96.56) (91.03) (89.51) (84.98)(102.57) (67.74) Med 253.35 305.65 357.20 354.20 302.00 266.00 297.10Min 227.10 153.70 185.60 208.10 184.60 197.00 249.20 Max 464.40 431.70396.20 447.50 412.70 398.80 345.00

TABLE 18 WBC, ANC, and Platelet Counts (K/CU MM) INH + RMP Baseline Day4 Day 8 Day 11 Day 15 Day 28 Day 42 WBC (K/cu mm) n 8 8 8 8 8 7 4 Mean(SD) 9.86 (3.47) 8.22 (4.01) 8.75 (5.16) 8.28 (3.83) 7.80 (4.36) 8.19(3.50) 8.36 (5.61) Med 8.59 6.95 6.40 5.60 5.85 6.69 6.10 Min 6.76 4.084.71 5.27 4.27 4.90 4.67 Max 15.84 16.90 20.23 16.40 15.73 14.20 16.55ANC (K/cu mm) n 8 8 8 8 8 7 4 Mean (SD) 6.71 (3.69) 4.97 (4.37) 5.45(5.14) 5.04 (4.26) 4.53 (4.10) 5.09 (3.83) 5.50 (5.38) Med 5.21 3.333.26 3.22 2.74 3.44 3.82 Min 3.00 1.78 1.36 1.73 1.47 1.59 1.18 Max13.37 14.69 16.98 14.26 12.42 11.41 13.18 Platelets (K/cu mm) n 8 8 8 88 6 3 Mean (SD) 363.16 (148.71) 376.78 (217.40) 429.23 (194.32) 416.00(190.23) 365.20 (163.83) 387.13 (287.01) 427.90 (234.40) Med 336.80320.00 358.30 372.45 300.30 245.80 490.00 Min 194.30 136.20 233.30185.00 222.00 175.00 168.70 Max 580.30 785.00 746.00 750.00 671.60874.70 625.00

TABLE 19 WBC, ANC, and Platelet Counts (K/CU MM) - INH + 10 mg-RifalazilBaseline Day 4 Day 8 Day 11 Day 15 Day 28 Day 42 WBC (K/cu mm) n 8 8 8 88 4 3 Mean (SD) 7.97 (1.41) 7.85 (2.66) 8.36 (3.43) 7.96 (2.78) 8.22(2.60) 6.05 (2.69) 5.87 (1.18) Med 7.73 7.90 8.61 8.20 7.95 5.82 5.98Min 6.59 5.20 4.47 3.75 4.61 3.01 4.64 Max 11.24 13.41 15.07 12.29 12.099.57 7.00 ANC (K/cu mm) n 8 8 8 8 8 4 3 Mean (SD) 4.91 (1.13) 4.61(1.95) 4.98 (2.57) 4.71 (2.34) 4.51 (2.46) 3.27 (1.73) 3.08 (1.33) Med4.94 4.10 5.09 4.76 4.22 3.53 2.65 Min 3.33 2.60 1.67 1.46 1.42 0.932.02 Max 6.70 7.99 8.86 7.80 7.89 5.09 4.58 Platelets (K/cu mm) n 8 8 88 8 3 2 Mean (SD) 358.01 (98.58) 391.89 (100.83) 403.21 (104.39) 358.35(95.30) 344.36 (53.48) 335.67 (94.42) 262.30 (48.55) Med 333.25 377.60408.65 376.90 341.30 300.10 262.30 Min 293.00 277.7 273.10 222.20 271.00264.20 227.90 Max 590.00 577.30 569.00 463.00 426.40 442.70 296.70

TABLE 20 WBC, ANC, and Platelet Counts (K/CU MM) INH + 25 mg-RifalazilBaseline Day 4 Day 8 Day 11 Day 15 Day 28 Day 42 WBC (K/cu mm) n 7 7 7 77 3 2 Mean (SD) 11.67 (6.27) 10.49 (6.79) 11.76 (7.15) 8.76 (4.86) 8.86(4.49) 9.64 (5.59) 11.59 (6.19) Med 10.37 8.05 10.24 7.42 7.62 9.4311.59 Min 6.46 4.63 4.08 3.70 3.62 4.16 7.21 Max 24.23 22.69 21.68 17.2315.79 15.33 15.97 ANC (K/cu mm) n 7 7 7 7 7 3 2 Mean (SD) 8.52 (5.17)7.07 (6.52) 8.90 (6.45) 5.91 (4.42) 5.97 (3.84) 6.01 (3.64) 7.39 (3.77)Med 6.62 4.05 7.78 4.40 5.17 6.70 7.39 Min 4.01 2.85 2.28 2.03 1.70 2.074.72 Max 18.70 17.06 18.69 14.57 12.81 9.25 10.05 Platelets (K/cu mm) n7 7 7 7 7 4 3 Mean (SD) 395.03 (244.00) 273.60 (143.15) 426.57 (258.92)416.89 (248.05) 373.04 (146.65) 307.63 (141.68) 251.37 (147.00) Med319.60 222.80 407.50 461.20 410.90 314.10 185.40 Min 159.10 117.00165.80 141.00 154.50 136.50 148.90 Max 846.20 483.30 930.00 884.90547.00 485.80 419.80

Table 21 summarizes the plasma concentrations data of rifalazil measuredin patients that received rifalazil at zero hour. The data are separatedinto 2 groups and are identified as INH+10 mg rifalazil (Group 3) andINH+25 mg rifalazil (Group 4). The concentration of rifalazil in plasmais presented in ng/mL and are shown as a timecourse (hours and days)wherein values were determined at several times over the two weeks ofthe study.

TABLE 21 Rifalazil Concentration in Plasma (ng/mL) Hour Day 8 Day 8Treatment Group 0 3 6 9 12 24 48 72 H-0 H-6 Day 11 Day 15 INH + 10 mgKRM n 4 4 4 4 4 4 2 3 3 4 2 3 Mean 0 9.7 12.58 9.95 10.7 3.6 2.5 1.4 016.83 2.4 0 (SD) (0) (9.97) (7.92) (8.50) (1.68) (1.12) (0.42) (1.22)(0) (5.04) (0.57) (0) Med 0 9.0 11.9 10.3 11.4 3.55 2.5 2.0 0 18.0 2.4 0Min 0 0 4.2 0 6.2 2.3 2.2 0 0 9.7 2.0 0 Max 0 20.8 72.7 19.2 11.6 5.02.8 2.2 0 21.5 2.3 0 INH + 25 mg KRM n 6 6 6 6 6 6 5 4 5 6 6 4 Mean 015.93 28.47 21.62 16.83 7.72 3.48 2.15 0 29.02 4.12 0.85 (SD) (0)(13.90) (12.53) (10.34) (4.16) (3.03) (2.12) (1.47) (0) (12.01) (2.48)(1.7) Med 0 11.4 30.85 22.15 18.1 6.7 3.9 2.7 0 28.5 3.0 0 Min 0 2.8 5.59.2 11.1 4.3 0 0 0 11.7 2.2 0 Max 0 38.4 42.7 38.2 20.6 12.2 5.3 3.2 043.0 6.3 3.4

The observed plasma levels of rifalazil were similar to those seen innormal volunteers. Table 21 shows that the plasma concentration ofrifalazil increases from the zero level to 9.7 ng/mL for 10 mg ofrifalazil and to 15.93 ng/mL in three hours showing a maximumconcentration of the drug in plasma at six hours following the drugadministration (12.68 ng/mL for 10 mg rifalazil and 28.47 ng/mL for 25mg rifalazil). The drug concentration in plasma slowly decreases butthere is still measurable amount of drug in plasma at 72 hours (3 days).After the second administration at day 8, the drug concentrations inboth groups were found to be higher than following the firstadministration.

The data obtained in TB patients show that rifalazil administered onceor twice weekly is effective for treatment of tuberculosis and haslesser adverse reactions than other currently available treatments.Additionally, these data once again illustrate the unexpected differencebetween human and animal pharmacokinetic data.

C. Comparison of Rifalazil Treatment with Rifampin and Rifabutin

When similar studies with two other currently FDA approved rifamycins,rifampin and rifabutin were made, the following adverse reactions wereobserved with typical daily dose of rifampin and rifabutin.

Typical daily dose of rifampin >600 mg given daily has resulted in thefollowing adverse reactions including “flu-like” symptoms, such asfever, headaches, chills, malaise and other reactions listed above inhematopoietic reactions, such as leukopenia, thrombocytopenia, acutehemolytic anemia, and in cutaneous, gastrointestinal (GI) and hepaticreactions, shortness of breath, shock and renal failure.

GI reactions included heartburn, epigastric distress, anorexia, nausea,vomiting, gas, cramps, diarrhea, sore mouth and tongue, pseudomembranouscolitis, pancreatitis, and were experienced by 1% to 2% subjects.

Hepatic reactions included asymptomatic elevations of liver enzymes (upto 14%) and hepatitis (<1%). Other reactions such as shock-like syndromewith hepatic involvement were present but rare. Abnormal liver functiontests, transient abnormalities in liver function tests, such aselevations in serum bilirubin, BSP, alkaline phosphatase, serumtransaminase were also observed.

Dermatologic reactions included rash observed in 1% to 5% of subjects,pruritus, urticaria, pemphigoid reaction, flushing.

CNS reactions included headache, drowsiness, fatigue, dizziness,inability to concentrate, mental confusion, generalized numbness,behavioral changes, and, rarely, also ocular myopathy.

Hematologic reactions included eosinophilia, transient leukopenia,hemolytic anemia, decreased hemoglobin, hemolysis and thrombocytopenia.

Musculoskeletal reactions included ataxia, muscular weakness, pain inextremities, osteomalacia and myopathy.

Opthalmologic reactions included visual disturbances and exudativeconjunctivitis.

Renal reactions included hemoglobinuria, hematuria; renal insufficiencyand acute renal failure.

Miscellaneous other adverse reactions associated with rifampin includedmenstrual disturbances, fever, elevation in BUN and elevated serum uricacid, possible immunosuppression, isolated reports of abnormal growth oflung tumors, reduced 25-hydroxycholecalciferol levels, edema of face andextremities, shortness of breath, wheezing, decrease in blood pressureand shock.

Rifabutin (150-300 mg single dose) also causes adverse reactionsalthough it is generally better tolerated than rifampin. Discontinuationof therapy due to an adverse event was required in 16% of patientsreceiving rifabutin vs. 8% with placebo. Primary reasons fordiscontinuation were rash adverse reactions included flu-like symptoms,hepatitis, hemolysis, arthralgia, myositis, chest pressure or pain withdyspnea, skin discoloration (<1%), seizure, paraesthesia, aphasia,confusion, and non-specific T wave changes on ECG. When rifabutin wasadministered at doses from 1050 to 2400 mg/day, generalized arthralgiaand uveitis occurred. These experiences abated when rifabutin wasdiscontinued.

The incidence of neutropenia in patients treated with rifabutin wassignificantly greater than in patients treated with placebo. Althoughthrombocytopenia was not significantly more common amongrifabutin-treated patients, in rare cases rifabutin has been clearlylinked to thrombocytopenia. One patient developed thromboticthrombocytopenic purpura, which was attributed to rifabutin.

Both rifampin and rifabutin are FDA approved drugs for treatment oftuberculosis. In view of the high dosages required to suppress M.tuberculosis and in view of the severe a and numerous adverse reactionsassociated with administration of these drugs, there clearly is a needfor new drugs and methods of treatment for tuberculosis.

Rifalazil has been shown to have antibacterial activity againstMycobacterium tuberculosis, Mycobacterium avium, Chlamydia pneumoniae,H. pylori and other bacteria. Despite the adverse reactions described inthe clinical trials 001-004, the novel method for treatment oftuberculosis with rifalazil administered once or twice-a-week is amethod of choice. It effectively lowers CFU in TB patients whenadministered in well tolerated doses (10 and 25 mg/dose) once ortwice-a-week for 4-62 weeks. At these dosages, the adverse reactions areat most mild or not observable.

Both the animal studies and studies on human volunteers suggest thatrifalazil has fewer side effects than rifampin, and rifabutin and hashigher anti-bacterial activity, especially against Mycobacteriumtuberculosis, Mycobacterium avium, Chlamydia pneumoniae and H. pylori.

III. Administration and Formulation

The drug product is administered systemically or parenterally, i.e.orally, intravenously, by aerosol, by suppositories or in any otheracceptable pharmaceutical form.

Rifalazil may be formulated and administered as standalone drug withvarious pharmaceutically acceptable additives and excipients, or incombination with other drugs such isoniazid, ethambutol, streptomycin,pyrazinamide, capreomycin, ethionamide, cycloserine, kanamycin,tobramycin or amikacin, with other rifamycins, that is, it can be alsocombined with rifampin or rifabutin, with other antibacterial agents,analgesics, antitussives, etc. Various ratios of these drugs to eachother will depend on the intended use, on the patients symptoms andseverity of the disease. Various combinations and ratios of drugs toeach other are within the skills of the pharmacist formulating therifalazil or rifalazil combination with other drugs.

Typically, the drug product will contain rifalazil, mannitol, USP;hydroxypropyl cellulose, NF; colloidal silicon, dioxide, NF; magnesiumstearate, NF; polysorbate 80, NF; and water in proportions that permitmaterial flow in capsule-filling equipment. For example, rifalazil willbe prepared in No. 3 hard gelatin dark blue opaque snap fit capsules, oras tablets, injectables, suppositories, etc.

For clinical studies described above, rifalazil capsules have beenprepared at several different strengths; 5 mg, 25 mg, 50 mg, and 100 mg.The drug in the 5 mg and 50 mg strength capsules has been blended withadditional mannitol (placebo) so that the capsules were equivalent inweight and therefore indistinguishable from the 100 mg and placebocapsules, appropriately packaged and labeled. The drug was stored atroom temperature.

EXAMPLE 1

Clinical Study Design—Healthy Volunteers

This example describes a design used for clinical studies describedabove. This design is a typical one. Dosages and drug administrationregimen was changed according to the aim of the study.

Study Design

Subjects in an open-label trial received a single or multiple dose of 5,25, 50 or 300 mg dose of rifalazil. The study was a randomized,double-blind, placebo-controlled intermittent dose study designed todetermine a maximum safe dosing regimen.

The subject population was divided into two treatment groups, eachconsisting of six subjects. Subjects in each group were randomized toreceive either placebo or rifalazil once weekly for 4 weeks with atwo-week follow-up period. The two treatment groups were separated by atleast two weeks to at allow sufficient time to review the safety data.Subjects in all groups received drug or placebo within 30 minutes ofeating a standard 60% fat breakfast.

Subjects in Treatment Group 1 were randomized to receive either a 25 mgdose (n=6), a 50 mg dose (n=6), or placebo (n=6) once a week (q.w.) fora total for four weeks.

Randomization and Blinding

Subjects were randomly assigned to and within a treatment group. Allstudies were blind or double blind.

Patient Selection

The study was conducted with healthy adult volunteers recruited from thepopulation at large.

Dose Selection

Dose selection for this study was based on the safety profile ofrifalazil obtained from three previous safety and pharmacokinetic (PK)studies. The results of these studies indicated that the incidence ofadverse reactions was greater following daily dosing than after a singledose and that the adverse reactions were more prolonged following dailydosing. Furthermore, there appeared to be a dose-dependent trend in theincidence of adverse reactions. A single 300 mg dose did produce moreadverse reactions than either the 30 mg or 100 mg single dose.Similarly, approximately twice as many adverse reactions were recordedfor the 25 mg daily dose group (003 trial) than for the 5 mg daily dosegroup and the adverse reactions were more prolonged than following asingle dose.

Drug Formulation

Rifalazil and matching placebo were prepared in No.3 hard gelatin darkblue opaque snap-fit capsules. Rifalazil capsules have been prepared atfour different strengths 5 mg, 25 mg, 50 mg and 100 mg. Rifalazil in the5 mg, 25 mg and 50 mg strength capsules has been blended with additionalmannitol (placebo) so that the capsules were equivalent in weight andtherefore indistinguishable from the placebo.

Dosage and Administration

All capsules were administered orally with water. Compliance wasassessed by visual examination of dosing and of the oral cavityfollowing administration.

EXAMPLE 2

Clinical Study Design for tuberculosis Patients

This example describes design used for clinical studies performed ontuberculosis patients.

The overall clinical studies and conditions were similar to thosedescribed in Example 1. Due to ethical considerations, there were noplacebo (control) patients in this study. All patients were treatedeither with 400 mg isoniazid daily, which is a typical and FDA approvedTB treatment, or with 400 mg isoniazid in combination with 600 mgrifampin daily, which is also typical and FDA approved TB treatment, orwith 400 mg isoniazid daily and 10 mg of rifalazil once-a-week, or with400 isoniazid daily and 25 mg of rifalazil once-a-week. Both latestregimens were experimental and performed under IND permit from FDA.

Results of the clinical trial 005 on tuberculosis patients are describedabove in Section II.B.

What is claimed:
 1. A method for treatment of bacterial infectionscaused by Mycobacterium tuberculosis, Mycobacterium avium complex,Chlamydia pneumoniae or Helicobacter pylori in human subjects byonce-a-week or twice-a-week administration of rifalazil in a dosage fromabout 1 to about 100 mg.
 2. The method of claim 1 wherein the dosage ofrifalazil is from 5 to 50 mg administered once-a-week or twice-a-week.3. The method of claim 2 wherein the dosage of rifalazil is from 10 to25 mg administered once-a-week or twice-a-week.
 4. The method of claim 3wherein the bacterial infection is caused by Mycobacterium tuberculosis.5. The method of claim 4 wherein the infection is tuberculosis.
 6. Themethod of claim 5 wherein the tuberculosis is treated by once-a-week ortwice-a-week administration of rifalazil for 4 to 62 weeks.
 7. Themethod of claim 6 wherein rifalazil is administered in combination withisoniazid, ethambutol, pyrazinamide, streptomycin, capreomycin,ethionamide, cycloserine, kanamycin, tobramycin or amikacin.
 8. Themethod of claim 7 wherein the tuberculosis is treated for 8 weeks. 9.The method of claim 7 wherein the tuberculosis is treated for 16-26weeks.
 10. The method of claim 1 wherein the infection is caused byChlamydia pneumoniae.
 11. The method of claim 10 wherein the Chlamydiapneumoniae infection is treated with once-a-week or twice-a-week dose ofrifalazil in dose from 1 to about 50 mg orally.
 12. The method of claim1 wherein the infection is caused by Mycobacterium avium complex. 13.The method of claim 12 wherein the Mycobacterium avium complex infectionis treated with once-a-week or twice-a-week dose of 1-50 mg of rifalazilalone or in combination with azithromycin or clanthromycin.
 14. Themethod of claim 1 wherein the rifalazil is administered orally,transdermally, parenterally, topically or by suppositories.
 15. Themethod of claim 14 wherein the topical administration is inhalation,buccal, or transdermal and wherein the parental administration isintravenous, intraperitoneal, subcutaneous or intramuscular.
 16. Themethod of claim 14 wherein the drug is administered orally in tablets,capsules, powders or as sustained release microcapsules.